The current invention relates to radioactively labeled compounds that find use in the visualization and analysis of compounds, particularly compounds containing or attached to a tetracysteine moiety.
Biarsenical compounds are known from, for example, Griffin et. al. Science 1998, 281, 269-272, Adams et. al. J. Am. Chem. Soc. 2002, 124, 6063-6076, Genin et. al. J. Am. Chem. Soc. 2008, 130, 8596-8597, Wilkins et. al. Bioorg. Med. Chem. Lett. 2009, 19, 4296-4298, Machleidt et. al. Meth. Mol. Biol. 2006, 356, 209-220, WO2007144077, WO2005040197, US20030083373, and WO03107010. These biarsenical compounds expose suitably located arsenic moieties. The specifically-located arsenic moieties bind strongly to tetracysteine sequences. Tetracysteine sequences are inherent features of some proteins. In other proteins, they may be inserted into the sequence, for example as a tag. A tetracysteine sequence may alternatively be present in a tag that associates non-covalently with a peptide or other compound.
The development of such biarsenical compounds was driven by an interest in the fields of molecular biology and medicine to enable visualisation of biologically relevant molecules by fluorescence tagging; for example for determining the location of a protein, or other biomolecule, within, for example, a cell or tissue.
Because the advent of biarsenical compounds was exclusively dependent on fluorescence for detection, biarsenicals are typically based on well established fluorescent compounds or cores, such as xanthene dyes including fluorones and fluoresceins. Such dyes have long been utilised in a variety of mapping and probing studies, all depending on the fluorescent properties of the dye, and may be prepared by methods such as those recently described in Jorge et. al. J. Org. Chem. 2005, 70, 6907-6912.
While biarsenical probes have been useful, their applicability is limited by the dependence on fluorescence detection. For example, organic dyes are well known to suffer from classical photobleaching, which constitutes an inherent drawback. Other aspects which hamper the extension of biarsenical probing include a relatively high detection limit (micromolar range) as well as poor penetration of the fluorescence signal (blue to red wavelength light), which precludes applications in complex matrices, such as in vivo. Despite these drawbacks, biarsenical compounds have been widely used and their binding to various protein structures has been widely studied.
US2003/0083373 is directed to the provision of biarsenical molecules, in particular fluorescein derivatives containing carboxyl groups. That document makes reference to carboxy-fluoroscein derivatives containing 3H or 125I. However, no synthesis details or data concerning radiolytic stability is provided for those compounds. US2003/0083373 exclusively provides synthesis details for biarsenicals by preparation from commercially available 2-arboxyphenylfluorones/fluoresceins.
The carboxylic acid group in such fluorescein analogues constitutes a site of high reactivity, and can interact with radiolabelling reagents. Any synthetic approach to such molecules would be complicated, likely requiring the use of protecting groups, having a negative impact on synthesis timeframe which is very critical in shortlived radiochemical synthesis. In fact, it is likely that the compounds mentioned in US2003/0083373 cannot be prepared using available radiochemical methodology.
A further drawback of such carboxy-fluorescein compounds is that they are subject to tautomerism, which greatly complicates synthesis, purification, and distribution in the study matrix of interest.